The species of Lactobacillus casei group, including Lb. casei, Lb. paracasei and Lb. rhamnosus, are generally recognized as safe (GRAS), and are widely used for commercial probiotics or as microorganisms for producing yogurt products. However, these species are taxonomically closely related. The genotypes and phenotypes of the species are very similar (the average similarity of the 16S rRNA genes among the species is >98.9%), so it is difficult to correctly distinguish species from each other. Furthermore, the functional characteristics of each probiotic are strain specific. Therefore, the Food and Agriculture Organization (FAO) and World Health Organization (WHO) of the United Nations announced in 2002 that it is essential to identify the scientific name of a probiotic to the strain level.
Pulsed-field gel electrophoresis (PFGE) is commonly used as the gold standard method for species discrimination and differentiation of different microorganisms. Although said method can achieve the purpose of intraspecies discrimination, it is complex and time-consuming. The reproducibility of the method is still a problem.
Multilocus sequence typing (MLST) is a more recent, highly developed taxonomic technique. Basically, MLST is established by sequencing 6-7 housekeeping genes having about 450 bp, and then aligning the sequences of these housekeeping genes to identify the mutated bases among the sequences so as to determine the sequence type (ST) of a microorganism. Since MLST provides good discrimination and reproducibility, and since it can be used to establish sequence information databanks and compare the sequences from different laboratories, MLST can not only improve the accuracy of PFGE but also replace it.
Cai et al. (“Genotypic and Phenotypic Characterization of Lactobacillus casei Strains Isolated from Different Ecological Niches Suggests Frequent Recombination and Niche Specificity.” Microbiology, 153, pp. 2655-2665, 2007) and Diancourt et al. (“Multilocus Sequence Typing of Lactobacillus casei Reveals a Clonal Population Structure with Low Levels of Homologous Recombination.” Applied and Environmental Microbiology, October 2007, pp. 6601-6611) used MLST to classify 40 and 52 strains of Lb. casei to 36 and 31 haplotypes, respectively. These and other publications have identified and selected many protein encoding genes, such as recA, yycH, dnaK, hsp60, tuf rpoA and pheS (preferably, recA, yycH, and pheS), as the markers for classifying Lb. casei. However, most of these identifying markers can only classify the microorganism to the species level, but cannot effectively determine different strains among the same species. In addition, MLST requires comparing and sequencing 6 to 7 gene loci so as to discriminate the bacterium. Therefore, MLST is also time-consuming and costly.
Alekseeva et al. (WO2013/100810 A2) used sequence alignment to compare the sequences of the fragments of MazEF and RelBE gene of the edible lactic acid bacteria, including Lb. acidophilus, Lb. helveticus, Lb. plantarum, Lb. paracasei, and Lb. rhamnosus to identify the inter- and intraspecies discriminations of the bacteria. The results of Alekseeva et al. show that the sequence similarity of the MazEF gene of Lb. paracasei and Lb. rhamnosus is about 83%, and thus these two bacteria can be distinguished from each other based on the gene. However, Alekseeva et al. did not include in the comparison the bacterium Lb. casei, which is closely related in taxonomy to those species. In addition, Alekseeva et al. did not use Lb. rhamnosus as a type strain for comparison. Therefore, the results of Alekseeva et al. cannot determine whether MazEF gene can be used to distinguish the interspecies relationship among Lb. casei, Lb. paracasei and Lb. rhamnosus. In the intraspecies discrimination, Alekseeva et al. found that through the MazEF gene analyses, 7 strains of Lb. paracasei can be classified into 3 haplotypes; and through the relE1 gene analyses, 5 strains of Lb. rhamnosus can be classified into 3 haplotypes.
Therefore, in this technical field, there is still a need to develop a method which can easily, quickly, simultaneously, and accurately identify the inter- and intraspecies relationships of the lactic acid bacteria.